Hemes are among the most prevalent and versatile cofactors in biological systems. One of the factors affecting the adaptability of these cofactors to a wide variety of different biological needs is the ease with which the redox state, and within each redox state, the electronic state of the iron, may be adjusted by porphyrin substituents and protein environment. During the next grant period we will concentrate on five projects: I. The structures, spectroscopic and redox properties of low-spin Fe(III) models of the cytochromes having large g max or Type I EPR spectra will be investigated by a wide array of spectroscopic techniques. At present we know that complexes with ligand plane dihedral angles as large as 30 degrees have Type II, while those with dihedral angles as small as 70 degrees have Type I EPR spectra. Detailed structural, spectroscopic and redox investigations of new complexes will allow us to determine how large the dihedral angles can be for Type II, and how small for Type I complexes. Electrochemical studies will allow investigation of the stabilities of the Fe(III) vs. Fe(II) axial ligand complexes, in order to see whether these complexes are indeed faithful models of the membrane-bound cytochromes having "large g max" EPA signals in the oxidized state. II. Several carefully-chosen ferriheme proteins and their mutants will be investigated by magnetic spectroscopic techniques (in particular, pulsed EPA) to determine the orientation of the axial histidine (or exogenous imidazole) ligands, in order to (1) determine whether the exogenous ligand complexes are faithful models of the membrane-bound cytochromes having "large g max" EPR signals in the oxidized state, and (2) to see if EPA signals can be assigned to particular hemes of complex multi-heme proteins. III. Models of the cytochromes, especially those having reduced heme rings and having axial or Type Ill EPA spectra suggestive of the (dxz,dyz)4 (dxy) ground state will be investigated by X-ray crystallography, magnetic spectroscopies and electrochemistry to determine whether they have this ground state, and if so, whether they have different redox reactivity than their (dxy)2(dxz,dyz)3 ground state counterparts. IV. The electronic ground state of model hemes having hydroperoxide as an axial ligand will be determined by pulsed EPA techniques, to determine whether they are (dxz,dyz)4(dxy)1 centers, and if so, V. H63M cytochrome b5, cytochrome P450cam, heme oxygenase and myoglobin having hydroperoxide as an axial ligand will be studied by pulsed EPA techniques to determine whether each is a (dxy)2(dxz,dyz)3 or (dxz,dyz)4(dxy)1 ground state center; if the latter, the mechanism of action of these enzymes will be better understood.